A tale of two synapses: the development of neurotransmitter phenotype in motor neurons. While it has been known for 60 years that motor neurons release acetylcholine at neuromuscular junctions, evidence has been accumulating that motor neurons also release an excitatory amino acid transmitter at synapses on other neurons. Several recent studies provide evidence that motor neurons release multiple neurotransmitters in a spatially segregated way between the distinct types of synapses they make - neuromuscular junctions in the periphery and inter- neuronal synapses in the spinal cord. Recently published work from our lab and others has shown that in cultures of motor neurons isolated from embryos and grown in the absence of contact with muscle cells, excitatory neurotransmission in the culture is entirely through glutamate with no detectable cholinergic neurotransmission. The recent availability of an optogenetic mouse model that allows reversible, real-time and cell-autonomous photo-activation of acetylcholine-releasing neurons provides an excellent tool to investigate the development of neurotransmitter phenotype in motor neurons. The objective of the proposed study is to use electrophysiology, confocal imaging and optogenetic approaches with motor neurons grown in culture and in spinal cord slices to determine the neurotransmitter phenotype of synapses formed by motor neurons on other neurons, and investigate how contact with muscle cells, the formation of neuromuscular junctions, and possibly acetylcholinesterase influence the neurotransmitter released. Insight into how neurons are guided to make different types of synapses on different targets, including developing spatially segregated release of multiple neurotransmitters, is crucial for understanding neuronal development. Motor neurons, which make two types of synapses clearly differentiated in structure, function and location, are an ideal model system in which to investigate differential synaptic development. Understanding how interaction with muscle completes the development of motor neurons will lead to insights into the pathophysiology of neuromuscular disorders including developmental motor neuron diseases such as spinal muscular atrophy and muscular dystrophy. In addition, as more researchers explore the use of differentiated pluripotent cells for potential replacement of motor neurons lost to disease or injury, a complete understanding of motor neuron differentiation and synaptic physiology will be crucial to advancing that research. This project is ideal for R15 support as Delaware State University is an Historically Black, predominantly undergraduate institution with approximately 80% African-American enrollment. Funding this research will increase opportunities for minority undergraduate and graduate students to directly experience the scientific process by taking part in important scientific research.